6. A cell line according to claim 5, wherein the cell line is HEK 293.

7. A method for production of a rFIX mutant according to claim 1,
comprising a) generating a nucleic acid encoding said rFIX mutant, b)
cloning the nucleic acid encoding said rFIX mutant, c) expressing the
nucleic acid encoding said rFIX mutant in a cell line, and d) purifying
said rFIX mutant.

8. A pharmaceutical composition comprising a FIX mutant according to
claim 1.

9. A method for treating a bleeding disorder associated with functional
defects of FVIII or deficiencies of FVIII, comprising the step of
administering a pharmaceutical composition according to claim 8 to a
patient in need thereof.

10. A method according to claim 9, wherein the bleeding disorder
associated with functional defects of FVIII or deficiencies of FVIII is
hemophilia A.

11. The method according to claim 9, wherein the bleeding disorder
associated with functional defects of FVIII or deficiencies of FVIII is
due to the development of FVIII inhibitor antibodies.

Description:

CROSS-REFERENCES TO RELATED APPLICATIONS

[0001] This application is a continuation of U.S. application Ser. No.
12/022,059, filed Jan. 29, 2008, which claims benefit of U.S. Provisional
Application No. 60/898,877, filed Feb. 1, 2007. The disclosure of each
application is hereby incorporated by reference in its entirety for all
purposes.

[0003] The blood coagulation cascade involves a series of serine protease
enzymes (zymogens) and protein cofactors. When required, an inactive
zymogen precursor is converted into the active form, which consequently
converts the next enzyme in the cascade.

[0004] The cascade is divided into three distinct segments: the intrinsic,
extrinsic, and common pathways (Schenone et al., Curr Opin Hematol.
2004;11:272-7). The activation of factor X (FX) is the common point of
the intrinsic and extrinsic pathways. The activation occurs either by the
extrinsic complex formed by activated factor VII (FVIIa) and tissue
factor, or by the intrinsic tenase complex composed of activated Factor
IXa (FIXa) and activated Factor VIIIa (FVIIIa) (Mann, Thromb. Haemostasis
1999;82:165-74).

[0005] Activated FX along with phospholipids, calcium, and factor Va (FVa)
then converts prothrombin to thrombin (prothrombinase complex), which in
turn cleaves fibrinogen to fibrin monomers. The monomers polymerize to
form fibrin strands. Factor XIIIa (FXIIIa) covalently bonds these strands
to one another to form a rigid mesh.

[0006] Deficiencies of the components of the intrinsic tenase complex,
FVIIIa and FIXa, lead to severe bleeding disorders, hemophilia A and B,
respectively. Hemophilia A is considered the classic form of hemophilia,
whereas hemophilia B is also known as Christmas disease. Hemophilia A and
B are the consequence of congenital deficiencies of FVIII and FIX,
respectively. The worldwide incidence of hemophilia A is approximately 1
case per 5,000 male individuals and of hemophilia B 1 case per 30,000.

[0007] Originally patients with severe hemophilia had a shortened lifespan
and diminished quality of life that was greatly affected by hemophilic
arthropathy. But life expectancy has increased from 11 years before the
1960s for patients who were severely affected to more than 50-60 years by
the early 1980s. This has been accomplished through the widespread use of
replacement therapy.

[0008] Nowadays the treatment of choice for the management of hemophilia A
is replacement therapy with various plasma derived or recombinant FVIII
concentrates. Although progress in the production of FVIII to ensure
purity, efficacy and viral safety has been made over the past decades,
some limitations remain. First of all, severe hemophilia A patients are
frequently affected by anti-FVIII inhibitor antibody formation, thus
rendering the therapy ineffective. Approximately 30% of patients with
severe hemophilia A develop alloantibody inhibitors that can neutralize
FVIII (Hay, Haemophilia 2006;12 Suppl 6:23-9, Peerlinck and Hermans,
Haemophilia 2006;12:579-90). Furthermore, acquired hemophilia may occur
which is the development of FVIII antibody inhibitors in persons without
a history of FVIII deficiency.

[0009] Attempts to overwhelm the inhibitors with large doses of human
FVIII have been tried. Also porcine FVIII which has low cross-reactivity
with human FVIII antibody has been administered. More frequently,
FVIII-bypassing agents, including FEIBA (factor eight inhibitor bypassing
activity), FIX complex and FVIIa have also been used.

[0010] Modification of the functional activity of the tenase complex would
also be an elegant approach to address several of the above discussed
issues in hemophilia treatment, i.e., deficiency of FVIII or FIX and
inhibitor development.

[0011] In the tenase complex FIXa has critical importance (Rawala-Sheikh
et al., Biochemistry 1990;29:2606-11). FIXa is a two-chain vitamin
K-dependent serine protease capable of hydrolysing the Arg194-Ile195
peptide bond in the FX molecule which leads to its activation
(Venkateswarlu et al., Biophys. J. 2002;82:1190-206). Although this
reaction can proceed slowly in solution, it is significantly accelerated
in the presence of negatively charged phospholipid surfaces. In vivo,
these surfaces are mainly provided by activated platelets and plasma
lipoproteins. The rate of the reaction is increased further by the
presence of FVIIIa.

[0012] FIXa exhibits very low catalytic activity in an in vitro system
lacking either the co-factor FVIIIa or the physiologic substrate FX. This
is in contrast to the closest related homologue, FXa, which shows
significant activity towards peptide substrates (in addition to its
physiologic substrate prothrombin), independent of its co-factor protein
FVa. Thus the drawback of the sophisticated regulation of this enzymatic
system is that failure of a single component such as FVIIIa or the
development of inhibitors suffices to interrupt the functional activity
of the tenase apparatus.

[0013] An improved FIX protein, which has improved FVIII-independent FX
activation potential could avoid this issue. Several amino acid residues
of FIXa are already known to be important for regulation of enzymatic
activity and interaction with both FVIIIa and FX.

[0014] The surface loop 99 of FIXa is important for regulation of FIXa
activity (Hopfner et al., Structure Fold Des. 1999;7:989-96). In the
non-complexed FIXa this loop is stabilized in an inactive conformation
and limits access of substrate to the catalytic machinery.

[0015] The mutations Y94F and K98T are located on the 99-loop, known to
contribute to FX substrate binding by forming of the recognition site of
the S2 and S4 pockets of FX. Y177F mimics the effect of activation by
FVIIIa. Tyrosin 177 locks the 99-loop in an inactive conformation, which
is released by binding of FVIIIa to FIXa (Sichler et al., J Biol Chem.
2003; 278:4121-26).

[0017] However, in none of these publications full length FIX mutants
expressed in mammalian cells showed an improved functional activity in a
meaningful activated partial thromboplastin time (aPTT) assay in
FVIII-depleted plasma or FVIII-inhibitor-patient plasma.

[0018] Thus, there remains a great need in the art for compositions and
methods that provide an improved FIX molecule that can be used for
treatment of patients with hemophilia A.

[0019] It was the inventive task of the present invention to develop novel
FIX proteins by introduction of amino acid exchanges, which have improved
FVIII-independent FX activation potential with coagulation FVIII activity
useful for the treatment of bleeding disorders.

BRIEF SUMMARY OF THE INVENTION

[0020] The present invention relates to recombinant blood coagulation
factor IX (rFIX) mutants having factor VIII (FVIII) independent factor X
(FX) activation potential. Five full length FIX proteins with novel
combinations of mutations of amino acids important for functional
activity of FIX, i.e. SEQ ID NO:4 (FIX-Y94F/K98T), SEQ ID NO:6
(FIX-Y94F/K98T/Y177F), SEQ ID NO:8 (FIX-Y94F/A95aK/K98T/Y177F), SEQ ID
NO:10 (FIX-Y94F/K98T/Y177F/I213V/E219G) and SEQ ID NO:12
(FIX-Y94F/A95aK/K98T/Y177F/I213V/E219G) and SEQ ID NO:2 (FIX wild type)
were cloned, expressed in HEK 293 and purified by a three step
purification protocol using anion exchange chromatography,
pseudo-affinity chromatography and affinity chromatography. Pre-activated
FIX was removed with biotinylated chloromethylketones and
streptavidine-sepharose. Among other assays the proteins were tested by
an activated partial thromboplastin time (aPTT) assay in FVIII-depleted
plasma as well as FVIII-inhibited patient plasma. In FVIII-depleted
plasma functional activity of a rFIX mutant was calculated as increased
FVIII equivalent activity. PdFIX and FIX-WT had no or only a minor FVIII
equivalent activity. From the 5 mutated proteins (at 5 μg/mL)
FIX-Y94F/K98T and FIX-Y94F/A95aK/K98T/Y177F/I213V/E219G had the greatest
effect with 14.7 and 16 FVIII equivalent mU/mL, and
FIX-Y94F/K98T/Y177F/I213V/E219G resulted in 12 FVIII equivalent mU/mL. In
FVIII-inhibited patient plasma the FEIBA equivalent activity was
calculated for analysis of FVIII independent FX activation potential.
PdFIX and FIX-WT had no or only a minor FEIBA equivalent activity. The
best rFIX mutant FIX-Y94F/A95aK/K98T/Y177F/I213V/E219G showed a FEIBA
equivalent activity of 162 mU/mL, FIX-Y94F/K98T and
FIX-Y94F/K98T/Y177F/I213V/E219G had both approximately 115 FEIBA
equivalent mU/mL. After pre-activation the rFIX proteins were tested in
FIX-depleted plasma containing inhibitors. At 1 μg/mL
FIXa-Y94F/K98T/Y177F/I213V/E219G displayed 73.4 times the activity of
pdFIXa, whereas FIXa-Y94F/A95aK/K98T/Y177F/I213V/E219G had a 17.1-fold
increased activity. Therefore the rFIX mutants can be used for the
treatment of bleeding disorder associated with functional defects of
FVIII, deficiencies of FVIII, or anti-FVIII inhibitor antibody formation.

BRIEF DESCRIPTION OF THE DRAWINGS

[0021]FIG. 1 shows the structure of the rFIX mutant cloning and
expression vector.

[0022]FIG. 2 shows a SDS-PAGE and Western Blot analysis of mutated rFIX
proteins.

[0028] The term "amino acid" within the scope of the present invention is
meant to include all naturally occurring L α-amino acids. The one
and three letter abbreviations for naturally occurring amino acids are
used herein (Lehninger, Biochemistry, 2d ed., Worth Publishers, New York,
1995: 71-92).

[0029] The rFIX mutant according to the present invention may be derived
from any vertebrate, e.g. a mammal.

[0030] According to the present invention, the term "FIX" does not
underlie a specific restriction and may include any FIX, with
heterologous or naturally occurring sequences, obtained via recombinant
DNA technology, or a biologically active derivative thereof Accordingly,
the term "rFIX mutant" includes any recombinant mutant derived from a FIX
protein sequence of any of the foregoing FIX. Accordingly, a FIX
polynucleotide or polypeptide sequence of the present invention is
typically derived from a mammalian FIX sequence including, but not
limited to, primate, e.g., human; rodent, e.g., rat, mouse, hamster; cow,
pig, horse, sheep, or any other mammalian sequence. In one specific
example of the present invention, the rFIX mutant is a recombinant mutant
of human FIX. Polynucleotide and polypeptide sequences of the FIX can be
found for example in the UniProtKB/Swiss-Prot Accession No. P00740. The
mutated rFIX of the invention may be a mutated full length or truncated
FIX. In a preferred embodiment of the present invention the mutated rFIX
has a full length sequence. In the present invention the chymotrypsinogen
numbering within the serine protease domain was used according to Hopfner
et al. (EMBO J. 1997;16:6626-35).

[0031] A wide variety of vectors can be used for the preparation of a rFIX
mutant according to the present invention and can be selected from
eukaryotic and prokaryotic expression vectors. Examples of vectors for
prokaryotic expression include plasmids such as pRSET, pET, pBAD, etc.,
wherein the promoters used in prokaryotic expression vectors include lac,
trc, trp, recA, araBAD, etc. Examples of vectors for eukaryotic
expression include: (i) for expression in yeast, vectors such as pAO,
pPIC, pYES, pMET, using promoters such as AOX1, GAP, GAL1, AUG1, etc;
(ii) for expression in insect cells, vectors such as pMT, pAc5, pIB,
pMIB, pBAC, etc., using promoters such as PH, p10, MT, Ac5, OpIE2, gp64,
polh, etc., and (iii) for expression in mammalian cells, vectors such as
pSVL, pCMV, pRc/RSV, pcDNA3, pBPV, etc., and vectors derived from viral
systems such as vaccinia virus, adeno-associated viruses, herpes viruses,
retroviruses, etc., using promoters such as CMV, SV40, EF-1, UbC, RSV,
ADV, BPV, and β-actin.

[0032] A mutated rFIX according to the present invention may be produced
by any method known in the art, for example any method applicable to
non-mutated rFIX. An example was first published by Kaufman et al. (J
Biol Chem. 1986;261:9622-8). An example of a commercially available rFIX
is BeneFIX® manufactured by Genetics Institute. The production of a
rFIX mutant may include any method for the generation of recombinant DNA
by genetic engineering, e.g. via reverse transcription of RNA and/or
amplification of DNA.

[0033] A nucleic acid sequence encoding a mutant rFIX protein according to
the invention may be generated by any method known in the art. Examples
are polymerase chain reaction (PCR) and cloning methods. In a preferred
embodiment of the present invention the DNA encoding a mutant protein of
the invention is generated by in vitro mutagenesis using specific primers
to generate the respective mutations.

[0034] Additionally, the recombinant DNA coding for a mutant rFIX
according to the present invention, e.g. a plasmid, may also contain a
DNA sequence encoding a selectable marker for selecting the cells which
have been successfully transfected with the plasmid. In an example of the
present invention, the plasmid may also confer resistance to a selectable
marker, e.g. to the antibiotic drug hygromycin, by delivering a
resistance gene, e.g. the hygromycin resistance gene conferring
resistance to the marker.

[0035] The production of a rFIX mutant may include any method known in the
art for the introduction of recombinant DNA into eukaryotic cells by
transfection, e.g. via electroporation or microinjection. For example,
the recombinant expression of human FIX mutant can be achieved by
introducing an expression plasmid containing the human FIX mutant
encoding DNA sequence under the control of one or more regulating
sequences such as a strong promoter, into a suitable host cell line by an
appropriate transfection method resulting in cells having the introduced
sequences stably integrated into the genome. The lipofection method is an
example of a transfection method which may be used according to the
present invention.

[0036] The production of a rFIX mutant may also include any method known
in the art for the cultivation of said transformed cells, e.g. in a
continuous or batchwise manner, and the expression of the rFIX mutant,
e.g. constitutive or upon induction. In one specific example of the
present invention the nucleic acid coding for rFIX mutant contained in
the host organism of the present invention is expressed via an expression
mode selected from the group consisting of induced, transient, and
permanent expression. Any expression system known in the art or
commercially available can be employed for the expression of a
recombinant nucleic acid encoding rFIX mutant, including the use of
regulatory systems such as suitable, e.g. controllable, promoters,
enhancers etc.

[0037] The production of a rFIX mutant may also include any method known
in the art for the isolation of the protein, e.g. from the culture medium
or by harvesting the transformed cells. For example, the rFIX
mutant-producing cells can be identified by isolating single-cell derived
populations, i.e. cell clones, via dilution after transfection and
optionally via addition of a selective drug to the medium. After
isolation the identified cell clones may be cultivated until confluency
in order to enable the measurement of the rFIX mutant content of the cell
culture supernatant by enzyme-linked immuno-sorbent assay (ELISA)
technique. Additionally, rFIX mutant secreted by the cells may be
identified for example by growing the cells in the absence of any growth
promoting fetal serum or components thereof. Vitamin K is added at
appropriate concentrations to improve the functional properties of the
rFIX mutant protein. After identification, high rFIX mutant producing
cell clones may for example be further propagated and/or stored via
cryopreservation. The rFIX mutant may be also co-expressed with vitamin K
reductase complex subunit 1 (VKORC1, Hallgren et al., Biochemistry
2006;45:5587-98) and/or furin (Wasley et al. J Biol Chem. 1993;268:
8458-65).

[0038] The host cell type according to the present invention may be any
eukaryotic cell. In a preferred embodiment the cell is a mammalian cell
with the ability to perform posttranslational modifications of rFIX
mutant. For example said mammalian cell is derived from a mammalian cell
line, like for example a cell line selected from the group consisting of
SkHep-, CHO-, HEK293-, and BHK-cells. In a specific example of the
present invention, the rFIX mutant is expressed in HEK293-derived cells.

[0039] There is no particular limitation to the media, reagents and
conditions used for culturing the cells in the cell culture of the
present invention including culturing the cells in a continuous or
batch-wise manner. The cells may be cultured also under serum-free or
serum- and protein-free conditions. In a specific example of the present
invention the cells are cultured in a mixture of Dulbecco's modified
Eagle's Medium and F-12 medium.

[0040] Additionally, the production of a rFIX mutant may include any
method known in the art for the purification of rFIX mutant, e.g. via
anion exchange chromatography or affinity chromatography. In one
preferred embodiment rFIX mutant can be purified from cell culture
supernatants by anion exchange chromatography, tandem-pseudoaffinity and
affinity chromatography. The purified rFIX mutant may be analyzed by
methods known in the art for analyzing recombinant proteins, e.g. the
ELISA technique and by electrophoresis techniques including
immuno-blotting.

[0041] The term "FVIII independent FX activation potential" as used herein
means the functional activity of a rFIX mutant of the present invention
and any other rFIX mutant protein which may be assessed for example by
measuring activated partial thromboplastin time (aPTT).

[0042] The aPTT assays represent meaningful assays for testing the
functional activity of a mutant rFIX protein because they measure the
clotting time in plasma. In principle the clotting activity of any
compound is determined by its addition to plasma samples and measurement
of time to clotting. This can be carried out for example in plasma
depleted with a protein or in plasma from inhibitor patients.

[0043] A variety of methods for an aPTT may be possible. In one preferred
embodiment of the present invention the aPTT is measured in FVIII
depleted plasma samples. The FVIII independent FX activation potential of
a FIX mutant may be calculated in FVIII-depleted plasma as increased
FVIII equivalent activity. PdFIX and FIX-WT usually have no or only a
minor FVIII equivalent activity (between 0 mU/mL and 1 mU/mL). Thus any
amino acid mutation leading to an increased FVIII equivalent activity as
compared to pdFIX or FIX-WT can be defined as increase. In a preferred
embodiment of the present invention the increased activity of a rFIX
mutant is at least 2 mU/mL, and more preferably more than 5 mU/mL.

[0044] In another preferred embodiment the FEIBA equivalent activity in
FVIII-inhibited patient plasma can be used for analysis of FVIII
independent FX activation potential. PdFIX and FIX-WT usually have no or
only a minor FEIBA equivalent activity (between 0 mU/mL and 15 mU/mL).
Any increase in FEIBA equivalent activity as compared to pdFIX or FIX-WT
can be defined as increase. In a preferred embodiment of the present
invention the increased activity is at least 30 mU/mL, and more
preferably more than 80 mU/mL.

[0045] In a further preferred embodiment the activity of a pre-activated
FIX mutant protein is determined in a clotting assay in FIX-depleted
plasma containing FVIII inhibitors. FIXa equivalent amounts can be
calculated from clotting times of a calibration curve made with pdFIXa.
In a preferred embodiment of the present invention the activity of a rFIX
mutant is increased at least 10 fold and more preferably 15 fold as
compared to pdFIX.

[0046] Another aspect of the present invention relates to a pharmaceutical
composition comprising a rFIX mutant having a FVIII independent FX
activation potential for treating a bleeding disorder associated with
functional defects of FVIII or deficiencies of FVIII.

[0047] The pharmaceutical composition may further comprise an auxiliary
agent, e.g. selected from the group consisting of a pharmaceutically
acceptable carrier, diluent, salt, buffer, or excepient. Said
pharmaceutical composition can be used for treating the above-defined
bleeding disorders. The pharmaceutical composition of the invention may
be a solution or a lyophilized product.

[0048] As used herein, the term "pharmaceutically acceptable" means
approved by a regulatory agency of US or EU government or listed in the
U.S. Pharmacopeia or other generally recognized pharmacopeia for use in
animals, and more particularly in humans.

[0049] It is another object of the present invention to provide a method
for treating a bleeding disorder associated with functional defects of
FVIII or deficiencies of FVIII comprising the step of administering a
pharmaceutical composition comprising a rFIX mutant having a FVIII
independent FX activation potential to a patient in need thereof.

[0050] The expression "bleeding disorder associated with functional
defects of FVIII or deficiencies of FVIII" as used herein includes
bleeding disorders, wherein the cause of the bleeding disorder may be
selected from the group consisting of a shortened in vivo-half-life of
FVIII, altered binding properties of FVIII, genetic defects of FVIII, and
a reduced plasma concentration of FVIII. Genetic defects of FVIII
comprise for example deletions, additions and/or substitution of bases in
the nucleotide sequence encoding FVIII whose absence, presence and/or
substitution, respectively, has a negative impact on the activity of
FVIII. FVIII inhibitor development may be also responsible for defects in
FVIII function. In one example of the present invention, the bleeding
disorder is hemophilia A.

[0051] The route of administration does not exhibit particular
limitations, and in one embodiment the protein of the present invention
may be administered by injection, such as intravenous, intramuscular, or
intraperitoneal injection. In a preferred embodiment of the present
invention the pharmaceutical composition may be administered
intravenously.

[0052] The present invention will be further illustrated in the following
examples, without any limitation thereto.

EXAMPLES

Example 1

Mutagenesis of FIX and Construction of FIX Expression Vectors

[0053] Publications referenced above discussing amino acid residues
important for the activation of FX by FIX and own considerations were
used for the construction of mutated FIX proteins. Two of the FIXa
mutations are located on the 99-loop, known to contribute to substrate
binding by forming the S2 and S4 substrate recognition site. The third
FIXa mutation, Y177T, is placed adjacent to the S4 site. Furthermore, in
FXa the 99-loop and 60-loop, both known to be highly involved in
substrate recognition, are stabilized by an inter-loop interaction
between the side chains of residues Y60 and K96, which might contribute
to the high amidolytic activity of FXa. Exchanging Ala-95a by Lys in FIXa
should yield in a salt bridge between A95aK and Glu-60 which might
influence the activity of FIXa similar to that of FXa. Finally five
FIX-mutants with different novel mutation combinations, i.e. SEQ ID NO:4
(FIX-Y94F/K98T), SEQ ID NO:6 (FIX-Y94F/K98T/Y177F), SEQ ID NO:8
(FIX-Y94F/A95aK/K98T/Y177F), SEQ ID NO:10
(FIX-Y94F/K98T/Y177F/I213V/E219G) and SEQ ID NO:12
(FIX-Y94F/A95aK/K98T/Y177F/I213V/E219G) were cloned in addition to SEQ ID
NO:2 (FIX-WT). The respective SEQ ID NOs for the encoding nucleic acids
are SEQ ID NO:3 (FIX-Y94F/K98T), SEQ ID NO:5 (FIX-Y94F/K98T/Y177F), SEQ
ID NO:7 (FIX-Y94F/A95aK/K98T/Y177F), SEQ ID NO:9
(FIX-Y94F/K98T/Y177F/I213V/E219G), SEQ ID NO:11
(FIX-Y94F/A95aK/K98T/Y177F/I213V/E219G), and SEQ ID NO:1 (FIX-WT).

[0054] For the construction of the rFIX plasmids the FVIII cDNA from
pCMVrFVIIIdB928/EDHPro (Herlitschka et al., J Biotechnol. 1998;61:165-73)
was replaced by human FIX cDNA. The FIX cDNA encodes a polymorphism of
human FIX leading to an amino acid exchange of Thr to Ala at position 194
in the activation peptide. The vector map of the plasmid is shown in FIG.
1. A schematic of the transcription unit, containing the human
cytomegalovirus (CMV) promoter/enhancer, the gene of interest (human FIX
cDNA), an internal ribosomal entry site (EMCV IRES), the selection
marker, the SV40 intron and the polyadenylation site is shown. The marker
is a chimeric construct, consisting of the wild-type dihydrofolate
reductase cDNA and the hygromycin phosphotransferase gene fused in frame.

[0055] For the construction of cDNA encoding FIX-Y94F/K98T,
FIX-Y94F/K98T/Y177F, FIX-Y94F/A95aK/K98T/Y177F,
FIX-Y94F/K98T/Y177F/I213V/E219G and FIX Y94F/A95aK/K98T/Y177F/I213V/E219G
site-directed mutagenesis was performed using the QuickChange
Site-Directed Mutagenesis Kit (Stratagene, La Jolla, Calif., USA). All
PCR reactions contained 125 ng sense primer, 125 ng antisense primer
(Invitrogen, Carlsbad, Calif., USA) and 5-50 ng dsDNA template, 2.5 units
of PfuTurbo DNA polymerase and dNTPs in a final volume of 50 μL
reaction buffer provided by the kit. After a pre-denaturation step of 1
minute at 95° C. PfuTurbo DNA Polymerase was added followed by 18
cycles of 95° C. for 30 seconds, 55° C. for 60 seconds and
68° C. for 12 minutes. The amplified product was incubated for 1
hour at 37° C. with DpnI to digest the methylated parental double
stranded DNA before transformation into XL1-Blue Supercompetent Cells.
For the construction of multiply mutated FIX cDNA this procedure was
repeated with the according primers (Invitrogen) as shown in Table 1. The
mutant FIX constructs were digested with restriction enzymes BsrGI and
XmaI (New England Biolabs, Ipswich, Mass., USA) and subsequently ligated
into the parental expression vector. Final FIX constructs were sequenced
(Applied Biosystems Model 373A Sequencer Applied Biosystems, Foster City,
Calif.) to confirm the mutations and were then linearized by AspEI for
transfection.

[0057] HEK 293 cells were grown in a mixture of Dulbecco's modified
Eagle's Medium and F-12 medium supplemented with 5% fetal calf serum.
Transfection was performed by lipofection using Lipofectamine® 2000
reagent (Invitrogen). One to 2 days before transfection HEK 293 cells
were seeded on 5 cm dishes to reach a confluence of 70-80%. On the day of
transfection the medium was exchanged 2 hours prior to the procedure. Six
μg of FIX cDNA were transfected according to the recommended
protocols. After 6 hours, fresh medium was added and the cells were
cultured for 1 to 2 days before passaging into 15 cm dishes and selection
of transfected cells with medium containing hygromycin at a concentration
of 200 μg/mL. Two to 3 weeks later, the surviving foci were isolated
into 24-well dishes in selective medium to produce stable cell lines.
Each clone was grown to confluence in the presence of 5 μg/mL vitamin
K1, and the secretion of FIX antigen into the medium was measured by an
ELISA. FIX secreted by high-producer clones was additionally assayed in
one-stage activated partial thromboplastin time assays (aPTT) and
visualized on Western blots.

[0058] The best cell lines were selected for large-scale production in
one-liter spinner flasks. Therefore cells were grown on 15 cm dishes to
90% confluence, trypsinized and counted in a CASY cell counter with a 150
μm capillary (Scharfe Systems, Reutlingen, Germany). 500 mL stirred
spinner flasks (60 rpm) were inoculated with 106 cells/mL in 200 mL
medium without fetal calf serum and supplemented with 5 μg/mL vitamin
K1 and 100 μg/mL hygromycin. The medium was expanded to a final volume
of 1000 mL over the next few weeks depended on the rate of growth of the
cells. The culture medium was collected twice weekly. Before storage at
-20° C. the culture medium was centrifuged and sterile filtrated
(GP EXPRESS PLUS Membrane, SCGPTO5RE, Millipore Corporation, Billerica,
Miss., USA) to remove cells and debris. The supernatant contained between
0.4 and 1 μg/mL rFIX antigen. rFIX WT produced 2.6 μg/mL.

[0059] FIX antigen levels were determined by a double antibody sandwich
ELISA. Therefore a sheep anti-human FIX affinity purified IgG (SAFIX-AP,
Affinity Biologicals Inc., Ancaster, ON, Canada) was diluted in
Tris-buffered saline (TBS, 25 mM Tris/HCl pH 7.4, 150 mM NaCl) to a
concentration of 2 μg/mL and dispensed in 100 μL aliquots into the
wells of a 96-well Nunc Maxisorp plate (Nunc, Roskilde, Denmark) which
was then kept at 4° C. over night. The plate was washed 3 times
with TBST (TBS+0.1% (v/v) Tween 20) followed by 1 hour blocking with 250
μL 3% non-fat dry milk powder (DMP) in TBS per well. The plate was
then washed and 100 μL of FIX-dilution in 1% DMP in TBST were
distributed in the wells. Serial dilutions of pdFIX (Enzyme Research
Laboratories, South Bend, Ind., USA) were used as standard protein. The
plate was incubated for 2 hours and then washed 5 times. Rabbit
anti-human FIX IgG (Accurate Chemical & Scientific Corp., Westbury, N.Y.,
USA) was diluted in TBST/1% DMP in a ratio of 1 to 6,000 and added to
each well in 100 μL aliquots for 1 hour. After 5 washing steps 100
μL of a goat anti-rabbit IgG (H+L) horseradish peroxidase
(HRP)-conjugate (Bio-Rad Laboratories, Hercules, Calif., USA) diluted 1
to 3,000 in TBST/1% DMP was added and incubated for 1 hour. Unbound
conjugated antibody was removed by washing the plate 5 times. The
addition of 100 μL 0.4 mg/mL o phenylenediamine (OPD, Sigma, St.
Louis, Mo., USA) and 0.4 mg/mL urea hydrogen peroxide in 50 mM
phosphate-citrate pH 5.0 started the color development. After an
incubation time of 7.5 min the reaction was stopped by the addition of 50
μL 0.5 N H2SO4. The absorbance at 492 nm was measured in an
ELISA reader (Labsystems iEMF Reader MF, Vantaa, Finland).

Example 3

Purification of Recombinant FIX Proteins

[0060] FIX proteins from serum-free conditioned medium were
ultrafiltrated, purified by anion exchange chromatography,
tandem-pseudoaffinity and affinity chromatography and polished by
inactivation and removal of preactivated rFIX. All purification steps
have been carried out on the chromatographic system Akta® Explorer 100
Air (Amersham Biosciences, Umea, Sweden) at 4° C.

[0061] The collected frozen serum-free culture medium from rFIX expression
was supplemented with 2 mM benzamidine and thawed at room temperature.
The pooled supernatants of each rFIX construct were concentrated on a
Sartorius UDF system using a 0.7 m2 polyvinylidene-difluorid (PVDF)
membrane with a 10 kDa molecular weight cut off. The system was run with
a flow of 330 mL/min.

[0065] The removal of preactivated rFIX was achieved by incubation of
rFIX-solutions with a fifteen fold molar excess of the two biotinylated
inhibitors
Biotinyl-ε-aminocaproyl-D-Phe-Pro-Arg-chloromethylketone (BFPRCK,
Bachem, Bubendorf, Switzerland) and
Biotinyl-Glu-Gly-Arg-chloromethylketone (BEGRCK, Haematologic
Technologies Inc., Essex Junction, Vt., USA) over night at 4° C.
FIX-Y94F/K98T was not treated with chloromethylketones.

[0066] rFIX fractions were supplemented with 0.1% ovalbumin and dialyzed
in a Slide-A-Lyzer MWCO 10 kDa (Pierce, Rockford, Ill., USA) against TBS
before streptavidin-sepharose (Amersham) was added in excess to the
chloromethylketones. Complexes of streptavidin-sepharose with
biotinylated rFIX-chloromethylketones were formed at 4° C. These
complexes were removed by 10 minute centrifugation at 4000 g and
4° C.

[0069] To analyze if binding of FVIIIa to a mutant FIXa protein can
neutralize the effect of the FIX mutations FX activation was also
measured in the presence of FVIIIa. Ten nM Recombinate Antihemophilic
Factor (Baxter, Thousand Oaks, Calif., USA) was incubated with FIXa and 4
minutes before the FX activation was started, and 10 nM thrombin (Enzyme

[0070] Research Laboratories, South Bend, Ind., USA) was added. FIXa
concentration was then 0.01 nM and the substrate was supplemented with 1
μM Pefabloc TH (Pentapharm, Basel, Switzerland) to prevent cleavage of
the substrate by thrombin. FXa formation was quantified as described
above by taking subsamples from 20 to 110 seconds.

[0071]FIG. 3 shows the FX activation by a FIXa protein (pdFIX, FIX-WT and
the 5 mutated proteins) in the absence (3A) and in the presence of FVIIIa
(3B). Apparent KM and kcat for FX activation without addition of FVIIIa
were then calculated and shown in Table 2.

[0072] As compared to pdFIX the double mutant FIX-Y94F/K98T showed a
two-fold increase whereas FIX-Y94F/K98T/Y177F/I213V/E219G and
FIX-Y94F/A95aK/K98T/Y177F/I213V/E219G enhanced the kcat by a factor of 17
and 6, respectively. FIX-WT and FIX-Y94F/K98T/Y177F activated FX at the
same rate as pdFIXa.

[0075] For experiments with FVIII depleted plasma (Dade Behring, Marburg,
Germany) serial dilutions of FVIII Immunate (Baxter) were used as
standards. FVIII inhibited patient plasma was from George King (Overland
Park, Kans., USA).

[0076] FIX proteins were first tested in FVIII-depleted plasma (FVIII
levels below 1%). Addition of FIX-WT and pdFIX to the plasma resulted in
no significant shortening of clotting time. However, all mutant FIX
proteins showed a concentration dependent decrease of clotting time. Five
μg/mL FIX proteins reduced the clotting time from 96 seconds to 64,
70, 67 and 64 seconds for FIX-Y94F/K98T, FIX-Y94F/K98T/Y177F,
FIX-Y94F/K98T/Y177F/I213V/E219G and
FIX-Y94F/A95aK/K98T/Y177F/I213V/E219G, respectively (FIG. 4A). Clotting
time of normal plasma (36 seconds) and that of FVIII-depleted plasma (96
seconds) are indicated by dotted lines. The FVIII Immunate standard
titration, fitted to a four-parameter algorithm, is shown on the lower
part of FIG. 4A. FVIII equivalent units (FIG. 4B) were calculated
according to the FVIII Immunate calibration (0.78-200 mU/mL).

[0077] From the 5 mutated proteins FIX-Y94F/K98T and
FIX-Y94F/A95aK/K98T/Y177F/I213V/E219G had the greatest effect with 14.7
and 16 FVIII equivalent mU/mL (Table 4), and the five-fold mutant
FIX-Y94F/K98T/Y177F/I213V/E219G resulted in 12 FVIII equivalent mU/mL.

[0078] An aPTT assay in FVIII-inhibited patient plasma is the most
relevant assay because it indicates for the function of the mutant FIX
proteins in Hemophilia A patients with FVIII inhibitors. Because FEIBA is
a possible treatment for these patients, reduced clotting times of FIX
proteins were compared to a standard curve of a FEIBA titration (0-1,000
mU/mL). One U/mL FEIBA restores the clotting time of normal blood in
inhibitor patient plasma (approximately 36 seconds). FIG. 5 shows the
results of the aPPT of pdFIX, FIX-WT and the 5 mutated proteins. Clotting
time of normal plasma (36 seconds) and that of FVIII inhibitor patient
plasma (142 seconds) are indicated by dotted lines. The FEIBA standard
titration, fitted to a 4-parameter algorithm, is shown on the lower part
of FIG. 5A. FEIBA equivalent units (FIG. 5B) were calculated according to
the FEIBA calibration (1.56-1,000 mU/mL).

[0079] The best mutant rFIX protein FIX-Y94F/A95aK/K98T/Y177F/I213V/E219G
showed a FEIBA equivalent activity of 162 mU/mL (Table 4). FIX-Y94F/K98T
and FIX-Y94F/K98T/Y177F/I213V/E219G had both activities of approximately
115 FEIBA equivalent mU/mL.

[0080] In the clotting assay described above FIX is directly activated by
FXIa before it can activate FX. A poor activity of a rFIXa mutant in the
clotting assay could therefore reflect impaired activation by FXIa or a
low activity in FX activation. To further investigate the FX activation
potential of the rFIX mutants without an influence of activation rates by
FXIa the clotting activity of the pre-activated rFIX mutants was
determined in clotting assays in FIX-depleted plasma containing FVIII
inhibitors. For activation pdFIX and rFIX mutants were diluted to 25
μg/mL in TBS containing 5 mM CaCl2 and 0.1% ovalbumin. FIX
activation was started by the addition of pdFXIa at a molar enzyme
substrate ratio of 1 to 500 at 37° C. FXIa was removed with
affinity purified goat anti-FXI IgG bound to protein G sepharose.

[0081] APTT was measured at concentrations of FIXa proteins between 0.0625
and 1 μg/mL. 50 μL FIX-depleted plasma containing FVIII-inhibitors
(goat anti FVIII, 150 BU/mL) and 50 μL of the respective activated FIX
proteins (0-1 μg/mL) were mixed with 50 μL aPTT-reagent for 1
minute at 37° C. Clotting time measurement was started by addition
of 50 μL 25 mM CaCl2. A titration with pdFIXa standard (0.0625-40
μg/mL), fitted to a four-parameter algorithm, is shown in black. Black
dotted lines show clotting times of FIX-depleted and FVIII-inhibited
plasma and of normal plasma.

[0082] 20 μg/mL pdFIXa restored clotting time to that of normal plasma.
All activated rFIXa proteins were more efficient than pdFIXa and reduced
clotting times in a concentration-dependent manner (FIG. 6A). To reach
the clotting time of normal plasma 1 μg/mL of
FIXa-Y94F/A95aK/K98T/Y177F/I213V/E219G and only 0.5 μg/mL
FIXa-Y94F/A95aK/K98T/Y177F were necessary. For a better comparison, FIXa
equivalent amounts were calculated from clotting times of a calibration
curve made with pdFIXa. At 1 μg/mL FIXa-Y94F/K98T/Y177F/I213V/E219G
displayed 73.4 times the activity of pdFIXa, whereas
FIXa-Y94F/A95aK/K98T/Y177F/I213V/E219G had a 17.1-fold increased activity
(FIG. 6B). Table 5 shows the pdFIXa equivalent activity given for 0.5
μg/mL of FIXa proteins.

[0083] This invention shows for the first time that a rationally designed
rFIX mutant can substitute for FVIII activity in both FVIII depleted and
FVIII inhibitor plasma. Therefore a rFIX mutant according to the present
invention can be used for treatment of a bleeding disorder associated
with functional defects of FVIII or deficiencies of FVIII and especially
as alternatives for bypassing agents for the treatment of FVIII inhibitor
patients.

[0084] It is understood that the examples and embodiments described herein
are for illustrative purposes only and that various modifications or
changes in light thereof will be suggested to persons skilled in the art
and are to be included within the spirit and purview of this application
and scope of the appended claims. All publications, accession numbers,
patents, and patent applications cited herein are hereby incorporated by
reference in their entirety for all purposes.